SAMPLE MANAGEMENT STANDARD OPERATING PROCEDURE RNase A Cleanup of DNA Samples Version Number: 1.0 Version 1.0 Date: 12/21/2016 Author(s): Yuko Yoshinaga, Eileen Dalin Reviewed/Revised by: Summary RNase A treatment is used for the removal of RNA from genomic DNA samples. RNase A cleaves the phosphodiester bond between the 5'-ribose of a nucleotide and the phosphate group attached to the 3'- ribose of an adjacent pyrimidine nucleotide. The resulting 2', 3'-cyclic phosphate is hydrolyzed to the corresponding 3'-nucleoside phosphate. Materials & Reagents Materials Vendor Stock Number Disposables 1.5 ml microcentrifuge tubes Reagents TE Buffer, pH 8.0 Ambion 9849 RNase A, DNase and protease-free (10 mg/ml) ThermoFisher EN0531 Sodium acetate buffer solution (3M, pH 5.2) VWR 567422 Ethanol, 200 proof Pharmco-Aaper 111000200CSPP 50x TAE Buffer Life Technologies/ Invitrogen MRGF-4210 SYBR® Safe DNA gel stain (10,000x concentrate in Life Technologies/ Invitrogen S33102 DMSO) DNA Molecular Weight Marker II Sigma 10236250001 Gel Loading Dye, Blue (6x) New England BioLabs B7021S Equipment Centrifuge 4°C Heat block 37°C Gel electrophoresis device Gel Imager Bio-Rad EH&S PPE Requirements: 1.1 Safety glasses, lab coat, and nitrile gloves should be worn at all times while performing work in the lab during this protocol. 1.2 Ethanol is a highly flammable and irritating to the eyes. Vapors may cause drowsiness and dizziness. Keep containers closed and keep away from sources of ignition such as smoking. 1 SAMPLE MANAGEMENT STANDARD OPERATING PROCEDURE Avoid contact with skin and eyes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Procedure 1. RNase A treatment 1.1. Top off the DNA volume to 200 ul in TE buffer (pH 8.0) 1.2. Add 10 ul of RNase A 1.3. Incubate 37°C for 1 hour 1.4. Check small aliquot (5 ul) on an agarose gel with no treatment control. Run gel 10-15 minutes. If there is no traceable of RNA, proceed to next step. If significant amounts of RNA are still present, add another 10 ul of RNase A and repeat the incubation 2. Ethanol Precipitation 2.1. Add 1/10 volume of 3 M sodium acetate to RNase A treated DNA 2.2. Add 2.5 volumes of 100% ethanol 2.3. Mix and spin down sample 2.4. Place at -80°C for 30 minutes (or -20°C for overnight) 2.5. Spin sample at 4°C for 20 minutes to pellet DNA 2.6. Carefully, pour off supernatant 2.7. Wash pellet with 70°C ethanol (cold) 2.8. Spin sample at 4°C for 3-5 minutes 2.9. Take out all ethanol with pipet tips 2.10. Air dry the pellet 2.11. Resuspend pellet with 100 ul (or appropriate volume) of TE 2.12. Quantify and qualify the DNA and store it at -80°C or -20°C Change History 2 .